The length and sequence of amino acids at the NS1-NS2A junction of the dengue virus polyprotein that are required for specific cleavage effected by the cis-acting function of NS2A identified by deletion analysis. Recombinant DNA sequences of NS1-NS2A, each containing a deletion in NS1 followed by a sequence of 3-20 amino acids at the C-terminus of NS1 preceding the cleavage site, were constructed and expressed using vaccinia virus as a vector. The NS1 product of recombinant vaccinia virus infected cells was immunoprecipitated and analyzed by SDS-polyacrylamide gel electrophoresis. The occurrence of cleavage between NS1-NS2A was indicated by the appearance of shortened NS1. Failure to cleave this site yielded a large NS1NS2A fusion protein. This analysis indicated that a minimum length of 8 amino acids at the NS1 C-terminus preceding the NS1-NS2A juncture is required for cleavage to take place.